BMP-10 COMPOSITIONS

Abstract: Purified BMP-10 proteins and processes for producing them are disclosed. DNA molecules encoding the BMP-10 proteins are also disclosed. The proteins may be used in the treatment of bone and cartilage defects and in wound healing and related tissue repair.

Description: TITLE OF THE INVENTION BMP-10 COMPOSITIONS The present invention relates to a novel family of purified proteins designated BMP-10, DNA encoding them, and processes for obtaining them. These proteins may be used to induce bone and/or cartilage formation and in wound healing and tissue repair. These proteins may also be used for augmenting the activity of other bone morphogenetic proteins. BACKGROUND OF THE INVENTION The search for the molecule or molecules responsible for the bone and cartilage-inductive activity present in bone and other tissue extracts has led to the discovery of a novel set of molecules called the Bone Morphogenetic Proteins (BMPs) . The structures of several proteins, designated BMP-1 through BMP-9, have previously been elucidated. The unique inductive activities of these proteins, along with their presence in bone, suggests that they are important regulators of bone repair processes, and may be involved in the normal maintenance of bone tissue. There is a need to identify whether additional proteins exist which play a role in these processes. The present invention relates to the identification of such a protein, which the inventors have designated BMP-10. SUMMARY OF THE INVENTION Bovine BMP-10 The bovine BMP-10 DNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) are set forth in the Sequence Listings. BMP-10 proteins are capable of inducing the formation of cartilage, bone or combinations thereof. BMP-10 proteins may be further characterized by the ability to demonstrate cartilage and/or bone formation activity in the rat bone formation assay described below. Bovine BMP-10 may be produced by culturing a cell transformed with a DNA sequence comprising nucleotide a DNA sequence encoding the mature BMP-10 polypeptide, comprising nucleotide #779 to nucleotide #1102 as shown in SEQ ID NO: 1, and recovering and purifying from the culture medium a protein characterized by the amino acid sequence comprising amino acids #1 to #108 as shown in SEQ ID NO:2 substantially free from other 94/26893 TPCT/US94/0529Q proteinaceo s materials with which iτ, is co-produced. For production in mammalian cells, the DNA sequence further comprises a DNA sequence encodinq a suitable propeptide 5' to and linked in frame to the nucleotide Gcq enαc encoding the mature BMP-10 polypeptide. The propeptide may be the native RMP-lo propeptide, or may be a propeptide from another protein of t-.he TGF-β superfamily. Human BMP-lo is expected to be homologous to bovine BMP-10- The invention, therefore, includes methods for obtaining the DNA sequences encoding hitman BMP-10, the DNA sequences obtained by those methods, and the human protein encoded by those DNA sequences. This method entails utilizing the bovine BMP-10 nucleotide sequence or portions thereof to design probes to screen libraries for the human gene or coding sequences or fragments thereof using standard techniques. A DNA sequence encoding part of the human BMP-10 pro ' -gβin (SEQ ID Nθj3) and the corresponding afflino acid sequence (SEQ ID NO:4) are set forth herein. These sequences piay also be used in order to design probes to obtain the complete human BMP-lo gene or coding sequences through standard techniques- Human BMP-10 may be produced by culturing a cell transformed with the BMP-10 DNA sequence and recovering and purifying BMP-10 from the culture medium. The purified expressed protein is substantially free from other protejnaceous materials with which it is co-produced, as well as from other contaminants. The recovered purified protein is contemplated to exhibit cartilage and/or bone formation activity. The proteins l ^of the invention may be further character zed by the ability to demonstrate cartilage and/or bone formation activity in the rat bone formation assay des^rib^d below. Another aspect of the invention provides pharmaceutical compositions contain g a therapeutically effective amount of a BMP-10 protein in pharmaceutically acceptable vehicle o carrier. BMP-10 compositions of the invention may be used i the formation of cartilage. These cowpositions may further be utilized for the formation of bone.. BMP-J-0 compositions ma also be used for wound healing and tissue repair. Compositions of the invention may further include at least one other therapeutically useful agent such as the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for instance in United States Patents 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication O91/18098; and BMP-9, disclosed in PCT publication O93/00432. The compositions of the invention may comprise, in addition to a BMP-10 protein, other therapeutically useful agents including growth factors such as epidermal growth factor (EGF) , fibroblast growth factor (FGF) , transforming growth factor (TGF- α and TGF-/3) , and insulin-like growth factor (IGF) . The compositions may also include an appropriate matrix for instance, for supporting the composition and providing a surface for bone,,and/or cartilage growth. The matrix may provide slow release of the osteoinductive protein and/or the appropriate environment for presentation thereof. The BMP-10 compositions may be employed in methods for treating a number of bone and/or cartilage defects, periodontal disease and various types of wounds. These methods, according to the invention, entail administering to a patient needing such bone and/or cartilage formation wound healing or tissue repair, an effective amount of a BMP-10 protein. These methods may also entail the administration of a protein of the invention in conjunction with at least one of the novel BMP proteins disclosed in the co-owned applications described above. In addition, these methods may also include the administration of a BMP-10 protein with other growth factors including EGF, FGF, TGF-α, TGF-/3, and IGF. Still a further aspect of the invention are DNA sequences coding for expression of a BMP-10 protein. Such sequences include the sequence of nucleotides in a 5' to 3' direction illustrated in SEQ ID NO: 1 or SEQ ID NO: 10, DNA sequences which, but for the degeneracy of the genetic code, are identical to the DNA sequence SEQ ID NO: 1 or SEQ ID NO: 10, and encode the protein of SEQ ID NO: 2 or SEQ ID NO: 11. Further included in the present invention are DNA sequences which hybridize under stringent conditions with the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 10 and encode a protein having the ability to induce the formation of cartilage and/or bone. Preferred DNA sequences include those which hybridize under stringent conditions [see, T. Maniatis et al. Molecular Cloning (A Laboratory Manual) . Cold Spring Harbor Laboratory (1982), pages 387 to 389]. Finally, allelic or other variations of the sequences of SEQ ID NO: 1 or SEQ ID NO: 10, whether such nucleotide changes result in changes in the peptide sequence or not, but where the peptide sequence still has BMP-10 activity, are also included in the present invention. A further aspect of the invention includes vectors comprising a DNA sequence as described above in operative association with an expression control sequence therefor. These vectors may be employed in a novel process for producing a BMP- 10 protein of the invention in which a cell line transformed with a DNA sequence encoding a BMP-10 protein in operative association with an expression control sequence therefor, is cultured in a suitable culture medium and a BMP-10 protein is recovered and purified therefrom. This process may employ a number of known cells both prokaryotic and eukaryotic as host cells for expression of the polypeptide. The vectors may be used in gene therapy applications. In such use, the vectors may be transfected into the cells of a patient in vitro, and the cells may be reintroduced into a patient. Alternatively, the vectors may be introduced into a patient n vivo through targeted transfection. Descripton of the Sequences SEQ ID NO:l is the nucleotide sequence encoding part of the bovine BMP-10, derived from clone λ7r-20. SEQ ID NO:2 is the amino acid sequence containing the mature bovine BMP-10 polypeptide. SEQ ID NO:3 is a partial nucleotide sequence of human BMP- 10. SEQ ID NO:4 is a partial amino acid sequence for human BMP- 10 polypeptide. SEQ ID NO:5 and 6 are primers to bovine BMP-10 used to isolate the human BMP-10 or other BMP-10 proteins. SEQ ID NO:7 is a DNA sequence that is inserted into pMT2 CXM to add an Xhol recognition site near the SV40 origin of replication. SEQ ID NO:8 is a DNA sequence inserted into pMT21 to insert an Xhol recognition site upstream from the DHFR gene. SEQ ID NO:9 is a DNA sequence comprising a portion of the EMC virus leader sequence. SEQ ID NO: 10 is a DNA sequence encoding the complete human BMP-10 protein, including the complete propeptide at nucleotides # 160 to # 1107, and the mature polypeptide at nucleotides # 1108 to # 1431, derived from the cDNA clone HFL-3 and the genomic clone 20GEN.3. SEQ ID NO: 11 is the amino acid sequence encoded by SEQ ID N0:10. Detailed Descripton of the Invention BMP-10 The bovine BMP-10 nucleotide sequence (SEQ ID NO: 1) and encoded amino acid sequence (SEQ ID NO: 2) are set forth in the Sequence listings herein. The coding sequence of the mature bovine BMP-10 protein begins at nucleotide #779 and continues through nucleotide #1102. Purified bovine BMP-10 proteins of the present invention are produced by culturing a host cell transformed wth a DNA sequence comprising the DNA coding sequence of SEQ ID NO: 1 from nucleotide #167 to #1102, or from nucleotide #779 to #1102, and recovering and purifying from the culture medium a protein which contains the amino acid sequence or a substantially homologous sequence as represented by amino acids #-204 to #108 or # 1 to # 108 of SEQ ID NO: 2. A host cell may be transformed with a coding sequence encoding a propeptide suitable for the secretion of proteins by the host cell is linked in proper reading frame to the coding sequence for the mature BMP-10 protein. For example, see United States Patent 5,168,150, the disclosure of which is hereby incorporated by reference, in which a DNA encoding a precursor portion of a mammalian protein other than BMP-2 is fused to the DNA encoding a mature BMP-2 protein. Thus, the present invention includes ohimerir: DNA molecules comprising a DNA sequence encoding a propeptide from a member of the TGF-/3 superfa ily of proteins, other than BWP-10, is linked in correct reading frame to a DNA sequence encoding a BMF-10 polypeptide. The term "chimeric" is used to signify that the propeptide originates from a different polypeptide than the BMP-10. The human BMP-10 sequence of the present invention is obtained using the whole or fragments erf the bovine BMP-iO DNA sequence, or the partial human BMP-10-sequence of SEQ ID NO:3, as a probe. Thus, the human BMP-10 DNA -sequence—comprise the DNA sequence of nucleotides #30 to #167 of SEQ ID NO: 3. This partial sequence of the human BMP-10 DNA sequence corresponds WP.Π to nucleotides #899 to #1036 of the bovine BMP-10 P A sequence shown in SEQ ID NO: 1. The human BMP-10 protein comprises the sequence of amino acids #1- #46 of SEQ ID NO: 4. It is expected that BMP-10, as expressed _by mammalian cells such as CHO cells, exists as a heterogeneous population of active species of BMP-10 protein with varying N-termini. It is expected that active species will - co rise " " an ami o ac±ύ sequence beqinning with the cysteine residue at amino acid #7 of SEQ TD NO:l, or will comprise additional amino acid sequence further in the N-terminal direction. Thus, it is expected that DNA sequences encoding active BMP-10 proteins will comprise a nucleotide sequence com is g nucleotides -#779 or #797 to # 1102 of SEQ ID NO:l or nucleotides # 1108 or # 1126 to # 1431 of SEQ ID NO: 10. The N-terminus of human BMP-10 has been experimentally determined by expression in E. coli to be as follows: [M]NAKGNYX RTPLYIDFKEI, wherein X designates an amino acid residue, with no clear signal, which is consistent with a cysteine residue at that location. Thus, it appears that the N- ter inus of this species of BMP-10 is-at amino acid #1 of SEQ ID NQ;i or SEQ ID NO:10, and a DNA sequence encoding said species Of BMP-10 would om ris nucleotides #779 to 11D2 of SEQ ID N0:1 or #1108 to 1431 of SEQ ID NO:10. The -apparent molecul-ar weight of human Activin WC monomer was determined by SDS-PAGE to be approximately 10-12 kd on a Novex 16% triσine gel. Molecular weight of monomer by electrospray ionization mass spectrometry is 12292.5 on a Finnigan TSQ 7000. The human BMP-10 protein exists as a clear, colorless solution in 0.1% trifluoroacetic acid. The BMP-10 proteins recovered from the culture medium are purified by isolating them from other proteinaceous materials from which they are co-produced and from other contaminants present. BMP-10 proteins may be characterized by the ability to induce the formation of cartilage and/or bone, for example, in the rat bone formation assay described below. The BMP-10 proteins provided herein also include factors encoded by the sequences similar to those of SEQ ID NO: 1, but into which modifications are naturally provided (e.g. allelic variations in the nucleotide sequence which may result in amino acid changes in the polypeptide) or deliberately engineered. For example, synthetic polypeptides may wholly or partially duplicate continuous sequences of the amino acid residues of SEQ ID NO: 2. These sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with bone growth factor polypeptides of SEQ ID NO: 2 may possess bone growth factor biological properties in common therewith. Thus, they may be employed as biologically active substitutes for naturally-occurring BMP-10 and other BMP- 10 polypeptides in therapeutic processes. Other specific mutations of the sequences of BMP-10 proteins described herein involve modifications of glycosylation sites. These modifications may involve O-lin ed or N-linked glycosylation sites. For instance, the absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at asparagine-linked glycosylation recognition sites. The asparagine-linked glycosylation recognition sites comprise tripeptide sequences which are specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either asparagine-X- threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence. Additionally, bacterial expression of BMP-10 protein will also result in production of a non-glycosylated protein, even if the glycosylation sites are left unmodified. The present invention also encompasses the novel DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and coding for expression of BMP-10 proteins. These DNA sequences include those depicted in SEQ ID NO: 1 in a 5' to 3' direction and those sequences which hybridize thereto under stringent hybridization conditions [for example, 0.1X SSC, 0.1% SDS at 65°C; see, T. Maniatis et al, Molecular Cloning (h Laboratory Manual) . Cold Spring Harbor Laboratory (1982), pages 387 to 389] and encode a protein having cartilage and/or bone inducing activity. These DNA sequences also include those which comprise the DNA sequence of SEQ ID NO: 3 and those which hybridize thereto under stringent hybridization conditions and encode a protein having cartilage and/or bone inducing activity. Similarly, DNA sequences which code for BMP-10 proteins coded for by the sequences of SEQ ID NO: 1, or BMP-10 proteins which comprise the amino acid sequence of SEQ ID NO: 2, but which differ in codon sequence due to the degeneracies of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) also encode the novel factors described herein. Variations in the DNA sequences of SEQ ID NO: 1) or SEQ ID NO: 3 which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded are also encompassed in the invention. Another aspect of the present invention provides a novel method for producing BMP-10 proteins. The method of the present invention involves culturing a suitable cell line, which has been transformed with a DNA sequence encoding a BMP-10 protein of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the BMP- 10 proteins recovered and purified from the culture medium. The purified proteins are substantially free from other proteins with which they are co-produced as well as from other contaminants. Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) . The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature. 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol.. 5.(7) :1750-1759 (1985) or Howley et al, U.S. Patent 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples, is the monkey COS-1 cell line. The mammalian cell CV-l may also be suitable. Bacterial cells may also be suitable hosts. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis. Pseudomonas , other bacilli and the like may also be employed in this method. For expression of the protein in bacterial cells, DNA encoding the propeptide of BMP- 10 may not be necessary. Many strains of yeast cells known to those skilled in the art may also be available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8.:277-298 (Plenum Press 1986) and references cited therein. Another aspect of the present invention provides vectors for use in the method of expression of these novel BMP-10 polypeptides. Preferably the vectors contain the full novel DNA sequences described above which encode the novel factors of the invention. Additionally, the vectors contain appropriate expression control sequences permitting expression of the BMP-10 protein sequences. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the present invention. Additionally, the sequence of SEQ I NO:i or other sequences «xιt;σdinιj BMP—10 ptoteinε could b manipulated to express a mature BMP-10 protein by deleting BMP 10 encoding propeptide sequences and replacing them wit 5 sequences encoding the complete ptopeptides of other BM proteins or members of the TGF-β superfamily. Thus, the presen invention includes chimeric NA molecules encoding a propeptid from member of the TGF-β supertamily linked in correct readin frame to a DNA seq ence εnσodinq a BMP-io polypeptide. η n The vectors may be employed in the method of transformin cell lines and contain selected -regulatory—seq ces -i operative association with the DNA coding—seq ences -of th invention which are capable of directing the replication an expression thereof in selected host cells. Regulatory sequence 15 for such vectors are known to those skilled in the art and ma be selected depending upon hp host cells. Such selection i routine and does not form part of the present invention. A protein of the present invention, " which induce cartilage and/or bone formation in circumstances where bone i 20 not normally formed, has application in th -heaiing ^~ σ£ bon fractures ap cartilage defects in humans and other animals Such a preparation employing a BMP-10 protein may hav prophylactic use in closed a? well as open fracture reductio and also in the improved fixation of artificial joints. JDe_nojr 25 bone formation induced by an esteogeniσ agent contributes to th repair of congenital, trauma induced, or oncologic resectio induced craniofacial defects, and also is useful in cosmeti plastic surgery. A BMP-10 protein may be_U5ied in the treatmen of periodontal disease, and in other tooth repair processes 30 Such agents may provide an environment-to"^at ract ~ bone-formin cells, stimulate growth of bone-forming cells or induc differentiation of progenitors oir bone-forming call*. BMP-i polypeptides of the invention may-— lso be useful in th treatment of osteoporosis, A —variety of " osteogenic 35 car lage-indxiσing and bone inducing- factors —have bee described * See, e.g., European a en -applications 148,155 an 169,016 for discussions thereof. The proteins of the invention may also be used in wound healing and related tissue repair. The types of wounds include, but are not limited to burns, incisions and ulcers. (See, e.g. PCT Publication W084/01106 for discussion of wound healing and related tissue repair) . A further aspect of the invention is a therapeutic method and composition for repairing fractures and other conditions related to cartilage and/or bone defects or periodontal dis¬ eases. The invention further comprises therapeutic methods and compositions for wound healing and tissue repair. Such compositions comprise a therapeutically effective amount of at least one of the BMP-10 proteins of the invention in admixture with a pharmaceutically acceptable vehicle, carrier or matrix. It is expected that the proteins of the invention may act in concert with or perhaps synergistically with other related proteins and growth factors. Further therapeutic methods and compositions of the invention therefore comprise a therapeutic amount of at least one BMP-10 protein of the invention with a therapeutic amount of at least one of the other BMP proteins disclosed in co-owned applications described above. Such combinations may comprise separate molecules of the BMP proteins or heteromolecules comprised of different BMP moieties. For example, a method and composition of the invention may comprise a disulfide linked dimer comprising a BMP-10 protein subunit and a subunit from one of the "BMP" proteins described above. Thus, the present invention includes a purified BMP-10 polypeptide which is a heterodimer wherein one subunit comprises at least the amino acid sequence from amino acid #1 to amino acid #108 of SEQ ID NO:2, and one subunit comprises an amino acid sequence for a bone morphogenetic protein selected from the group consisting Of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9. A further embodiment may comprise a heterodimer of BMP-10 moieties. Further, BMP-10 proteins may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF) , fibroblast growth factor (FGF) , platelet derived growth factor (PDGF) , transforming growth factors (TGF-α and TGF-/3) , and k-fibroblast growth factor (kFGF) , parathyroid hormone (PTH) , leukemia inhibitory factor (LIF/HILDA/DIA) , insulin-like growth factors (IGF-I and IGF-II) . Portions of these agents may also be used in compositions of the present invention. The preparation and formulation of such physiologically acceptable protein compositions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art. The therapeutic compositions are also presently valuable for veterinary applications due to the lack of species specificity in BMP proteins. Particularly domestic animals and thoroughbred horses in addition to humans are desired patients for such treatment with the BMP-10 proteins of the present invention. The therapeutic method includes administering the composition topically, systemically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than the BMP-10 proteins which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the BMP composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition includes a matrix capable of delivering BMP-10 or other BMP proteins to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. The matrix may provide slow release of BMP-10 and/or other bone inductive protein, as well as proper presentation and appropriate environment for cellular infiltration. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the BMP-10 compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides. Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The biocera ics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. The dosage regimen will be determined by the attending physician considering various factors which modify the action of the BMP-10 protein, e.g. amount of bone weight desired to be formed, the site of bone damage, the condition of the damaged bone, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and the types of BMP proteins in the composition. The addition of other known growth factors, such as IGF I (insulin like growth factor I) , to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of bone growth and/or repair. The progress can be monitored, for example, x-rays, histomorphometric determinations and tetracycline labeling. The following examples illustrate practice of the present invention in recovering and characterizing bovine BMP-10 protein a d employing it to recover the human and other BMP-10 proteins, obtaining the human proteins and expressing the proteins via t .iiHrtbinant techniques EXAMPLE 1 Bovine BMP-tQ 800,000 recombinants of a bovine σenomic librar constructed in the vector XEMBL3 are ÷ r tΛl at a-ϊensity-of 800 recombinant bacteriophage plaques per plate on 100 plates. Duplicate nitrocellxilose replicas of the recombinan bacteriophage plaques are m de from these plates and amplified, A fraqment of human BMP-7 DNA corresponding to nucleotides #108 to #1403 (Figure 4, United States Patent 5,141,905) is 52 P labelled by the random priming procedure of Feinberg et al. [Anal. Biocheia. 132: 6-13 (1983)] a d hybridized to one set o filters in standard hybidization buffer (5X SSC, 0.1% SDS, 5 Denhardt's, 100 μg/ l salmon sperm DNA) f-SHB) at S0°C for 2 to days. The filters are washed under- reduce stringenc conditions (4X SSC, 0.1% SDS at 60* .) . Multiple ^positivel hybridizing recombinants are noted. " 5 positively hybridizin recombinant baσteriophage plaques are selected and replated fo secondaries. Duplicate nitrocellulose replicas of th recombinant plagues are made from these 52 secondary plates an amplified. One set of nitrocellulose filters is hybridised to th um^π BMP-7 DNA probe as described above and washed under th same reduced stringency conditions. The other se of filters i hybridized to a mixed BMP-5, BMP-6, and BMP-7 probe in SHB a >5 ύ C overnight and washed with a 0.IX SSC, 0.1% SDS at G5" (stringent hybridization and wash conditions) . The mixed prob consists of relatively equal amount:s - of 32 P-labelled DN fragments comprising nucleotides #1452 to #2QS0 (Fig e -4 United States Patent 5,106,748) of the -human BMP-5 sequence nucleotides #1395 to #1698 (Figure- , United States Paten 5,187,076) of the human BMP-6 sequence-r-and nucleotides #1081 t #1 01 (Figure 4, United States patent 5,141,905) of the huma BMP-7 sequence. The BMP-5, BMP-6 and BMP-7 DNA fragments ar 32 P-labelled by the random priming procedure and equal numbers o counts per minute (cpms) of each probe are combined and added to the SHB containing the other set of nitrocellulose filter replicas of the 52 secondary plates. Fourteen recombinants, which hybridized positively to the human BMP-7 probe under the reduced stringency conditions and exhibited weak or no hybridization to the mixed BMP-5/6/7 probe under high stringency conditions, are selected for further analysis. All 14 recombinants which exhibit these hybridization characteristics are plaque purified and bacteriophage DNA is prepared from each. The positively hybridizing region of one of the recombinants, designated λ7r-20, is localized to a 0.5 kb EcoRI/Hindlll restriction fragment. This fragment is subcloned into a plasmid vector (pGEM-3) and DNA sequence analysis is performed. The partial DNA sequence (SEQ ID NO: 1) and derived amino acid sequence (SEQ ID NO: 2) of clone λ7r-20 are shown in the Sequence Listings. The bacteriophage λ7r-20 has been deposited with the American Type Culture Collection, 12301 Parkklawn Drive, Rockville, MD, 20852, on April 23, 1993 and has been given the accession number ATCC 75452. This deposit meets the requirements of the Budapest Treaty of the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures and regulations thereunder. This λ7r-20 clone encodes at least a portion of the bovine BMP-10 protein of the present invention. The nucleotide sequence of clone λ7r-20 contains an open reading frame of at least 938 bp, as defined by nucleotides #165 to #1102 of SEQ ID NO:l (#165-166 being the last two thirds of a codon which is interrupted by an intron) . The open reading frame encodes at least 312 amino acids of a BMP-10 protein. The encoded 312 amino acid BMP-10 protein includes the full mature bovine BMP-10 protein (amino acid #1 to #108 of SEQ ID NO:2) , as well as the C-terminal portion of the propeptide region of the primary translation product (amino acid #-204 to #-1 of SEQ ID N0:2). A consensus splice acceptor sequence immediately preceding the BMP-10 coding sequence at #165 to #1102, and an in frame stop codon at position #101 to #103, suggests the presence of intron sequences in the 5' direction of nucleotide #165. Based upon the knowledge of other BMP proteins and other proteins within the TGF-/3 family, it is predicted that the precursor polypeptide would be cleaved at the multibasic sequence ARG-ILE-ARG-ARG in agreement with a proposed consensus proteolytic processing sequence of ARG-X-X-ARG. Cleavage of the BMP-10 precursor polypeptide is expected to generate a 108 amino acid mature peptide beginning with the amino acid ASN at position #1. The processing of BMP-10 into the mature form is expected to involve dimerization and removal of the N-terminal region in a manner analogous to the processing of the related protein TGF-3 [Gentry et al., Molec. & Cell. Biol.. 8:4162 (1988); Derynck et al., Nature, 316:701(1985)]. It is contemplated therefore that the mature active species of BMP-10 comprises a homodimer of two polypeptide subunits, each subunit comprising amino acids #1 to #108 with a predicted molecular weight of approximately 12,000 daltons. Further active species are contemplated comprising amino acids #7 to #108, thereby including the first conserved cysteine residue. As with other members of the BMP and TGF-/3 family of proteins, the carboxy-terminal region of the BMP-10 protein exhibits greater sequence conservation than the more amino-terminal portion. The percent amino acid identity of the bovine BMP-10 protein in the cysteine-rich C-terminal domain (amino acids #7 to #108) to the corresponding region of other human BMP proteins and other proteins within the TGF-3 family is as follows: BMP- 2, 56%; BMP-3, 39%; BMP-4, 54%; BMP-5, 48%, BMP-6, 48%; BMP-7, 47%; BMP-8, 46%; BMP-9, 67%' Vgl, 50%; GDF-1, 40%; TGF-31, 37%; TGF-02, 37%; TGF-03, 37%; inhibin β(B) , 36%; inhibin 0(A) , 39%. EXAMPLE 2 Human BMP-10 Bovine and human osteoinductive factor genes are presumed to be significantly homologous, therefore the bovine coding sequence or a portion thereof is used as a probe to screen a human genomic library or as a probe to identify a human cell line or tissue which synthesizes the analogous human cartilage and/or bone protein. A human genomic library, such as Stratagene catalog #944201, may be screened with such a probe, and presumptive positives isolated and DNA sequence obtained. Evidence that this recombinant encodes a portion of the human BMP-10 relies of the bovine/human protein and gene structure homologies. Once a recombinant bacteriophage containing DNA encoding a portion of the human cartilage and/or bone inductive factor molecule is obtained, the human coding sequence can be used as a probe to identify a human cell line or tissue which synthesizes BMP-10 RNA. Alternatively, the bovine BMP-10 coding sequence can be used as a probe to identify such human cell line or tissue. Briefly described, RNA is extracted from a selected cell or tissue source and either electrophoresed on a formaldehyde agarose gel and transferred to nitrocellulose, or reacted with formaldehyde and spotted on nitrocellulose directly. The nitrocellulose is then hybridized to a probe derived from a coding sequence of the bovine or human BMP-10. Alternatively, the bovine BMP-10 coding sequence is used to design oligonucleotide primers which will specifically amplify a portion of the BMP-10 encoding sequence located in the region located between the primers utilized to perform the specific amplification reaction. It is contemplated that bovine and human BMP-10 sequences would allow one to specifically amplify corresponding human BMP-10 encoding sequences from mRNA, cDNA or genomic DNA templates. Once a positive source has been identified by one of the above described methods, mRNA is selected by oligo (dT) cellulose chromatography and cDNA is synthesized and cloned in λgtlO or other λ bacteriophage vectors known to those skilled in the art, for example, λ ZAP by established techniques (Toole et al., supra) . It is also possible to perform the oligonucleotide primer directed amplification reaction, described above, directly on a pre- established human cDNA or genomic library which has been cloned into a λ bacteriophage vector. In such cases, a library which yields a specifically amplified DNA product encoding a portion of human BMP-10 protein could be screened directly, utilizing the fragment of amplified BMP-10 encoding DNA as a probe. 94IZ-W ' PCT/US94/05290 Oligonucleotide- primers designed on the ba-sis of the DNA Rp iience of the bovine BMFiO genomic clone λ7r-20 are predicted tn allow the specific amplif cation of uman BMP-10 encodinq sequences. The following oligonucleotide-primer is designed on the basis of nucleotides #876 to #898 of the DNA sequence set forth in SEQ ID NO. 1 and synthesized on an automate DNA synthesi ZP.T . Primer A: TGCTCTAGACCTATGAATGTCGTGGTGTTTGC The first nine nucleotides of primer A (underlined) comprise the recognition sequence for the restriction cndonuclease Xb&T which can be utilized to facilitate the manipulation of a specifically amplified DNA sequence encoding the BMP-10 protein of the invention TΠ3 are thus not derived from he DNA sequence presented in SEQ ID NO: 1. The following oligonucleotide primer is -designed on the basis of nuclaotides #10SO to #1037 of the DNA sequence set forth in SEQ ID NO. 1 and synthesised on a automated DNA synthesizer-: Primer B: TAGGGAT.CCCTTGTAGGTGACGACGCCCTTATC The first nine nucleotides of primer B (underlined) comprise the recognition sequence for the restriction endonuclease Ba HI which can be utilized to facilitate the manipulation of a specifically amplified DNA sequence encoding the BMP-10 protein of the invention and ar-e thus not derived from the DNA sequence present in SEQ IDTSO: 1. The standard nucleotide symbols in the above identified primers are as follows? A, adenoεine; C, cytosine, G, guanine and T, thymine. Primers A and B identified above -are utilized-as-primers to allow the ampli ication of a specific nucleotide from human genomic DNA. the ampli ication reaction is performed as tollowε: Human genomic DNA (source: peripheral blood lymphocytes) is denatured at 100 ° c for five minutes- and on ice prior to addition to a reaction mixture containing 200 μM each deoxynucleotide triphosphates (dATP, -dGTP, dCTP and dTTP) 10 inM Tris-HCJ pH8.3, 50 rtiM KCl, 1.5 KiM MgCl 2 , 0.001% " gelatin, 1.25 units Taq DNA polymerase, 100 pM oligonucleotide primer A and 100 pM oligonucleotide primer B. This reaction mixture is then subjected to thermal cycling in the following manner: 3 minutes at 94°C, 1 minute at 50°C, 1 minute at 72°C for one cycle, then 1 minute at 94°C, 1 minute at 50°C, 1 minute at 72°C for thirty- nine cycles. The DNA which is specifically amplified by this reaction is separated from the excess oligonucleotide primers A and B utilized to initiate the amplification by the use of a DNA purification resin based protocol under the conditions suggested by the manufacturer. The resulting DNA product is digested with the restriction endonucleases Xbal and BamHI, phenol extracted, chloroform extracted. Buffer exchange and removal of small fragments of DNA resulting from the Xbal/BaHI restriction digest is accomplished by dilution of the digested DNA product in 10 mM Tris-HCl pH8.0, 1 mM EDTA followed by centrifugation through a centricon™ 30 microconcentrator (W.R. Grace & Co., Beverly, Ma.; Product #4209) . The resulting Xbal/BamHI digested amplified DNA product is subcloned into a plasmid vector (pBluescript) between the Xbal and BamHI restriction sites of the polylinker region. DNA sequence analysis of the resulting subclones indicates that the specifically amplified DNA sequence product encodes a portion of the human BMP-10 protein of this invention. The DNA sequence (SEQ ID NO. 3) and derived amino acid sequence (SEQ ID NO. 4) of this specifically amplified DNA fragment are set forth in the Sequence Listings. Nucleotides #1 to #29 of this sequence comprise a portion of oligonucleotide primer A and nucleotides #168 to #197 comprise a portion of oligonucleotide primer B utilized to perform the specific amplification reaction. Due to the function of oligonucleotide primers A and B (designed on the basis of bovine BMP-10 DNA seguence) in initiating the amplification reaction, they may not correspond exactly to the actual sequence encoding a human BMP-10 and are therefore not translated in the above amino acid sequence derivation. The DNA sequence, from nucleotide #30 to #167 of SEQ ID NO: 3, or portions thereof, specifically amplified from the human genomic DNA template can be utilized as a probe to identify additional human BMP-10 encoding sequences from human genomic or human cDNA libraries by standard hybridization/screening techniques known to those skilled in the art. Full-lenσth Human BMP-10 The full-length human BMP-10 DNA sequence (SEQ ID NO:10) and encoded amino acids sequence (SEQ ID NO:11) are described in the Sequence Listings. One million recombinants of a human fetal liver cDNA library (Clonetech catalog # HL 1064a) constructed in the vector λgtlO are plated at a density of 20,000 recombinant bacteriophge plaques per plate on 50 plates. Duplicate nitrocellulose replicas of the recombinant bacteriophage plaques are made from these plates. An oligonucleotide probe designed on the basis of nucleotides #85-#114 of SEQ ID NO:3 is synthesized on an automated DNA synthesizer. This oligonucleotide probe is radioactively labelled with γ 32 P-ATP and is hybridized to both sets of the duplicate nitrocellulose replicas in SHB at 65°C. Eleven positively hybridizing recombinants are noted. One of the positively hybridizing recombinants, named HFL-3,is plaque purified. Bacteriophage plate stocks of the purified HFL-3 cDNA clone are prepared and bacteriophage DNA is isolated. A bacteriophage stock of this cDNA clone has been deposited with the ATCC, 12301 Parklawn Drive, Rockville, Maryland USA under the requirements of the Budapest Treaty and designated as ATCC # . A portion of the DNA sequence of clone HFL-3 is set forth in SEQ ID NO: 10. One million recombinants of a human genomic library (Stratagene Catalog # 944201) constructed in the vector λFIX are plated at a density of 20,000 recombinant bacteriophge plaques per plate on 50 plates. Duplicate nitrocellulose replicas of the recombinant bacteriophage plaques are made from these plates. An oligonucleotide probe designed on the basis of nucleotides #355-#384 of SEQ ID NO:10 is synthesized on an automated DNA synthesizer. This oligonucleotide probe is radioactively labelled with 7 32 P-ATP and is hybridized to both sets of the duplicate nitrocellulose replicas in SHB at 65°C. Six positively hybridizing recombinants are noted. One of the positively hybridizing recombinants, named 20GEN.3,is plaque purified. Bacteriophage plate stocks of the purified 20GEN.3 genomic clone are prepared and bacteriophage DNA is isolated. A bacteriophage stock of this genomic clone has been deposited with the ATCC, 12301 Parklawn Drive, Rockville, Maryland USA under the reguirements of the Budapest Treaty and designated as ATCC # . A portion of the DNA sequence of clone 20GEN.3 is set forth in SEQ ID NO: 10. A portion of the DNA sequence of the genomic clone 20GEN.3 was determined to be identical to a portion of the DNA sequence of the cDNA clone HFL-3. The extent of this overlap (nucleotides #219-#316) of SEQ ID NO: 10 were used as a basis to compile the complete coding sequence of the BMP-10 protein. This sequence is presented in SEQ ID NO: 10 and it should be noted that nucleotides #1-#218 are derived entirely from the DNA sequence contained in genomic clone 20GEN.3 and nucleotides #317-#1584 are derived entirely from the DNA sequence contained in cDNA clone HFL-3, while nucleotides #219- #316 have been determined to be present in both 20GEN.3 and HFL- 3. SEQ ID NO:10 predicts a human BMP10 precursor protein of 424 amino acids. Based on the knowledge of other BMPs and other proteins within the TGF-β family, it is predicted that the precursor polypeptide would be cleaved at the multibasic sequence ARG-ILE-ARG-ARG (amino acids #-4 through #-1 of SEQ ID NO:11) in agreement with the proposed consensus proteolytic processing sequence ARG-X-X-ARG. Cleavage of the human BMP-10 precursor polypeptide at this location would generate a 108 amino acid mature peptide beginning with the amino acid ASN at position #1 of SEQ ID NO:11. The processing of human BMP-10 into the mature form is expected to involve dimerization and removal of the N-terminal region in a manner analogous to the processing of the related protein TGF-0 [L.E. Gentry, et al. Molec. & Cell. Biol. 8:4162 (1988); R.Derynck, et al., Nature 316:701 (1985) . It is contemplated that the mature active species of human BMP-10 comprises a homodimer of two polypeptide subunits, each subunit comprising amino acids #1-#108 of SEQ ID NO:11, with a predicted molecular weight of 12,000 daltons. WO 94/26S93 PCT/US94/Q529Q Further active species are contemplated comprising amino dt;ids #7-#108 thereby including the first conserved cysteine residue. Heterodi eric molecules comprising orie ~~ subunit of BMP-10 and another subunit of another member of the BMP/TGF»β-superfamily L> are also contemplated. Additional methods known to those-skilled in the art may be used to isolate other species' BMP-10 proteins of the invention. EXAMPLE 3 -?n BTQ SSAY3 10 A. Dnβgi ' -Jβtj n. of -2 Q C611S Use of the W-20 bone marrow stromal cells as an indicator call line i*ϊ b s d upon the conversion of these cells to oεteσblast-like cells after treatment V-it a BMP protein [Thies et al. Journal of Bone, and Mineral Research, 5:305 (1990); and j.5 Thies et al, Endocrinology, 130:1318 (E9-92) } _—Specifically, w- 20 cells are a clonal bone marrow stromal cell line derived from adult mice by researchers in the l boa o of Dr. D. Nathan, Children's Hospi l, Boston, MA. Treatment of W-20 calls with certain BMP proteins results in (l) increased—alkaline 20 p osphatase production, (2) induction of PTH stimulated cAMP, and (3) induction of osteocalein synthesis by the cells. While ( 1 ) and (2) represent characteristics associated with the osteoblast phenotype, the ability to synthesize osteocalein is a phenotypic property only displayed by mature osteoblasts, 2 5 Furthermore, to date we have observed conversion of W-2Q stromal cells to osteoblast-like cells only upon treatment with BMPs. In this manner, the in vitro activities-displayed by BMP treated W-20 Cells correlate with the __τ_ i^ g - ~ bone forming activity known for BMP3. 3 0 Below two in vitro assays useful in comparison of BMP activities of novel osteoinduct-ive molecules -are described. B. »20 Alkaline Phosphatase Assay Protocol W-20 cells are plated into 96 well tissue c lt ϊm pl-ates at a density of 10,000 cells per well in 200 ul-ef—media f ME with 35 10% heat inactivated fetal calf serum, 2 mM gluta ine and loo Units/ml penicillin + 100 μg/ml streptomycin. The cells are allowed to attach overnight in a 5%--a t-,—5% co 2 incubator at 37 °C . The 200 μl of media is removed from each well with a multichannel pipettor and replaced with an egual volume of test sample delivered in DME with 10% heat inactivated fetal calf serum, 2 mM glutamine and 1% penicillin-streptomycin. Test substances are assayed in triplicate. The test samples and standards are allowed a 24 hour incubation period with the W-20 indicator cells. After the 24 hours, plates are removed from the 37°C incubator and the test media are removed from the cells. The W-20 cell layers are washed 3 times with 200 μl per well of calcium/magnesium free phosphate buffered saline and these washes are discarded. 50 μl of glass distilled water is added to each well and the assay plates are then placed on a dry iσe/ethanol bath for quick freezing. Once frozen, the assay plates are removed from the dry ice/ethanol bath and thawed at 37°C. This step is repeated 2 more times for a total of 3 freeze-thaw procedures. Once complete, the membrane bound alkaline phosphatase is available for measurement. 50 μl of assay mix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl 2 , 5 mM p-nitrophenol phosphate, pH = 10.3) is added to each assay well and the assay plates are then incubated for 30 minutes at 37°C in a shaking waterbath at 60 oscillations per minute. At the end of the 30 minute incubation, the reaction is stopped by adding 100 μl of 0.2 N NaOH to each well and placing the assay plates on ice. The spectrophotometric absorbance for each well is read at a wavelength of 405 nanometers. These values are then compared to known standards to give an estimate of the alkaline phosphatase activity in each sample. For example, using known amounts of p-nitrophenol phosphate, absorbance values are generated. This is shown in Table I. Table I Absorbance values for known amounts of BMPs can be determined and converted to μmoles of p-nitrophenol phosphate cleaved per unit time as shown in Table II. Table II These values are then used to compare the activities of known amounts of BMP-10 to BMP-2. ~ - Osteocalein RIA Protocol W-20 cells are plated at lθ 6 cells per well in 24 well multiwell tissue culture dishes in 2 mis of DME containing 10% heat inactivated fetal calf serum, 2 mM glutamine. The cells are allowed to attach overnight in an atmosphere of 95% air 5% C0 2 at 37°C. The next day the medium is changed to DME containing 10% fetal calf serum, 2 mM glutamine and the test substance in a total volume of 2 ml. Each test substance is administered to triplicate wells. The test substances are incubated with the W- 20 cells for a total of 96 hours with replacement at 48 hours by the same test medias. At the end of 96 hours, 50 μl of the test media is removed from each well and assayed for osteocalein production using a radioimmunoassay for mouse osteocalein. The details of the assay are described in the kit manufactured by Biomedical Technologies Inc., 378 Page Street, Stoughton, MA 02072. Reagents for the assay are found as product numbers BT-431 (mouse osteocalein standard) , BT-432 (Goat anti-mouse Osteocalein) , BT-431R (iodinated mouse osteocalein) , BT-415 (normal goat serum) and BT-414 (donkey anti goat IgG) . The RIA for osteocalein synthesized by W-20 cells in response to BMP treatment is carried out as described in the protocol provided by the manufacturer. The values obtained for the test samples are compared to values for known standards of mouse osteocalein and to the amount of osteocalein produced by W-20 cells in response to challenge with known amounts of BMP-2. The values for BMP-2 94/26SW F T/US94/0529Q in d uced osteocalein synthesis by w-20 calls ± ~ shown in Table III. Table III Osteocalein Synthesis by-^W~20 Cells BMP-2 Concentration ng/ l Osteocalein Synthesis nqZ.we.ll ~ 0 0--S 4 D.8 R 2.2 16 2, 31 3, 62 ~ 5 , 125 6, 250 ~ s 500 9. 1000 10.0 ftGSEN MODIFIED. SAMPATH-REEPI ASSAY A modified version of the rat bone formation assay described in Sa path and Reddi, Proc. Natl. Acad. Sci. USA, 80, :6591-6595 (1983) is used to evaluate bone and/or cartilage activity of BMP proteins. T s modified assay is herein called the Rosen-modified sampath-Reddi — ssay, The ethanol precipitation step of the sampath-Reddi procedure is replaced by dialyzing (if the composition is a solution) or diafiltering (if the composition is a suspension) the -fraction tσ be ~ ssayed against water. The solution or suspension rs—then-eqαilibrated to 0.1% TFA. The resultinq solution is a " dded ~~ to 20- g-of rat matrix. A mock rat matrix sample not treatad-vith the protein serves as a control. This material s " -frozen and lyophilized and the resulting powder enclosed in #5 gelatin capsules. The capsules are implanted subcutaneously in the abdominal thoracic area of 21 -49 day old male T.ong Evans rats. The implants are removed after 7-14 days. Half of --each implant is used for a lk aline p h osphatase analysis [see, Reddi et al, Proc. Natl. ftgad- Sci., 6911601 (1972)], •b The other half of each implant is fixed and processed for histological analysis. 1 μm glycolmethacrylate sections are stained with Von Kossa and acid fuschin to score the amount of induced bone and cartilage formation present in each implant. The terms +1 through +5 represent the area of each histological section of an implant occupied by new bone and/or cartilage cells and matrix. A score of +5 indicates that greater than 50% of the implant is new bone and/or cartilage produced as a direct result of protein in the implant. A score of +4, +3, +2, and +1 would indicate that greater than 40%, 30%, 20% and 10% respectively of the implant contains new cartilage and/or bone. The BMP-10 proteins of this invention may be assessed for activity on this assay. EXAMPLE 5 Expression of BMP-10 In order to produce bovine, human or other mammalian BMP-10 proteins, the DNA encoding it is transferred into an appropriate expression vector and introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts by conventional genetic engineering techniques. The preferred expression system for biologically active recombinant human BMP- 10 is contemplated to be stably transformed mammalian cells. One skilled in the art can construct mammalian expression vectors by employing the seguence of SEQ ID NO: 1 or SEQ ID NO: 10, or other DNA seguences encoding BMP-10 proteins or other modified sequences and known vectors, such as pCD [Okayama et al., Mol. Cell Biol.. 2:161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO i., 4:645-653 (1985)] and pMT2 CXM. The mammalian expression vector pMT2 CXM is a derivative of p91023(b) (Wong et al.. Science 228:810-815. 1985) differing from the latter in that it contains the ampicillin resistance gene in place of the tetracycline resistance gene and further contains a Xhol site for insertion of cDNA clones. The functional elements of pMT2 CXM have been described (Kaufman, R.J., 1985, Proc. Natl. Acad. Sci. USA 82 . :689-693) and include the adenovirus VA genes, the SV40 origin of replication including the 72 bp enhancer, the adenovirus major late promoter including a 5' splice site and the majority of the adenovirus tripartite leader sequence present on adenovirus late mRNAs, a 3' splice acceptor site, a DHFR insert, the SV40 early polyadenylation site (SV40) , and pBR322 sequences needed for propagation in E_ t . coli. Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2- VWF, which has been deposited with the American Type Culture Collection (ATCC) , Rockville, MD (USA) under accession number ATCC 67122. EcoRI digestion excises the cDNA insert present in pMT2-VWF, yielding pMT2 in linear form which can be ligated and used to transform E. coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by conventional methods. pMT2 CXM is then constructed using loopout/in mutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). This removes bases 1075 to 1145 relative to the Hind III site near the SV40 origin of replication and enhancer sequences of pMT2. In addition it inserts the following sequence: 5' PO-CATGGGCAGCTCGAG-3' at nucleotide 1145. This sequence contains the recognition site for the restriction endonucleaεe Xho I. A derivative of pMT2CXM, termed pMT23, contains recognition sites for the restriction endonucleases PstI, Eco RI, Sail and Xhol. Plasmid pMT2 CXM and pMT23 DNA may be prepared by conventional methods. pEMC231 derived from pMT21 may also be suitable in practice of the invention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. As described above EcoRI digestion excises the cDNA insert present in pMT-VWF, yielding pMT2 in linear form which can be ligated and used to transform E ^ . Coli HB 101 or DH- 5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by conventional methods. pMT21 is derived from pMT2 through the following two modifications. First, 76 bp of the 5' untranslated region of the DHFR cDNA including a stretch of 19 G residues from G/C tailing for cDNA cloning is deleted. In this process, a Xhol site is inserted to obtain the following sequence immediately upstream from DHFR: 5' -CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3' PstI Eco RI Xhol Second, a unique Clal site is introduced by digestion with EcoRV and Xbal, treatment with Klenow fragment of DNA polymerase I, and ligation to a Clal linker (CATCGATG) . This deletes a 250 bp segment from the adenovirus associated RNA (VAI) region but does not interfere with VAI RNA gene expression or function. pMT21 is digested with EcoRI and Xhol, and used to derive the vector PEMC2B1. A portion of the EMCV leader is obtained from pMT2-ECATl [S.K. Jung, et al, J. Virol 62:1651-1660 (1989)] by digestion with Eco RI and PstI, resulting in a 2752 bp fragment. This fragment is digested with TaqI yielding an Eco RI-TaqI fragment of 508 bp which is purified by electrophoresis on low melting agarose gel. A 68 bp adapter and its complementary strand are synthesized with a 5' TaqI protruding end and a 3' Xhol protruding end which has the following sequence: 5'-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT TaqI GAAAAACACGATTGC-3' Xhol This sequence matches the EMC virus leader sequence from nucleotide 763 to 827. It also changes the ATG at position 10 within the EMC virus leader to an ATT and is followed by a Xhol site. A three way ligation of the pMT21 Eco Rl-Xhol fragment, the EMC virus EcoRI-TaqI fragment, and the 68 bp oligonucleotide adapter Taql-Xhol adapter resulting in the vector pEMC2Sl. This vector contains the SV40 origin of replication and enhancer, the adenovirus major late promoter, a cDNA copy of the majority of the adenovirus tripartite leader sequence, a small hybrid intervening sequence, an SV40 polyadenylation signal and the adenovirus VA I gene, DHFR and jS-lactamase markers and an EMC sequence, in appropriate relationships to direct the high level expression of the desired cDNA in mammalian cells. The construction of vectors may involve modification of the BMP-10 DNA sequences. For instance, BMP-10 cDNA can be modified by removing the non-coding nucleotides on the 5' and 3' ends of the coding region. The deleted non-coding nucleotides may or may not be replaced by other sequences known to be beneficial for expression. These vectors are transformed into appropriate host cells for expression of BMP-10 proteins. Additionally, the sequence of SEQ ID NO:l or SEQ ID NO: 10 or other sequences encoding BMP-10 proteins can be manipulated to express a mature BMP-10 protein by deleting BMP-10 encoding propeptide sequences and replacing them with sequences encoding the complete propeptides of other BMP proteins. One skilled in the art can manipulate the sequences of SEQ ID NO: 1 or SEQ ID NO: 10 by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by bacterial cells. For example, the coding sequences could be further manipulated (e.g. ligated to other known linkers or modified by deleting non-coding sequences therefrom or altering nucleotides therein by other known techniques) . The modified BMP-10 coding sequence could then be inserted into a known bacterial vector using procedures such as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA. 77:5230-5233 (1980). This exemplary bacterial vector could then be transformed into bacterial host cells and a BMP-10 protein expressed thereby. For a strategy for producing extracellular expression of BMP-10 proteins in bacterial cells, see, e.g. European patent application EPA 177,343. Similar manipulations can be performed for the construction of an insect vector [See, e.g. procedures described in published European patent application 155,476] for expression in insect cells. A yeast vector could also be constructed employing yeast regulatory sequences for intracellular or extracellular expression of the factors of the present invention by yeast cells. [See, e.g., procedures described in published PCT application WO86/00639 and European patent application EPA 123,289] . A method for producing high levels of a BMP-10 protein of the invention in mammalian cells may involve the construction of cells containing multiple copies of the heterologous BMP-10 gene. The heterologous gene is linked to an amplifiable marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells containing increased gene copies can be selected for propagation in increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.. 159:601-629 (1982) . This approach can be employed with a number of different cell types. For example, a plasmid containing a DNA sequence for a BMP- 10 of the invention in operative association with other plasmid sequences enabling expression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol.. 2:1304 (1982)] can be co-introduced into DHFR-deficient CHO cells, DUKX-BII, by various methods including calcium phosphate coprecipitation and transfection, electroporation or protoplast fusion. DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum, and subsequently selected for amplification by growth in increasing concentrations of MTX (e.g. sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al., Mol Cell Biol.. 5:1750 (1983). Transformants are cloned, and biologically active BMP-10 expression is monitored by the Rosen-modified Sampath-Reddi rat bone formation assay described above in Example 4. BMP-10 expression should increase with increasing levels of MTX resistance. BMP-10 polypeptides are characterized using standard techniques known in the art such as pulse labeling with [35S] methionine or cysteine and polyacrylamide gel electrophoresis. Similar procedures can be followed to produce other related BMP-10 proteins. EXAMPLE 6 Biological Activity of Expressed BMP-10 To measure the biological activity of the expressed BMP-10 proteins obtained in Example 5 above, the proteins are recovered from the cell culture and purified by isolating the BMP-10 proteins from other proteinaceous materials with which they are co-produced as well as from other contaminants. The purified protein may be assayed in accordance with the rat bone formation assay described in Example 4. Purification is carried out using standard techniques known to those skilled in the art. Protein analysis is conducted using standard techniques such as SDS-PAGE acrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley, et al. Anal. Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al. Proc. Natl. Acad. Sci. JJSA 7 :4350 (1979)] The foregoing descriptions detail presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descrip- tions. Those modifications and variations are believed to be encompassed within the claims appended hereto. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: GENETICS INSTITUTE, INC. (B) STREET: 87 CambridgePar Drive (C) CITY: Cambridge (D) STATE: Massachusetts (E) COUNTRY: USA (F) POSTAL CODE (ZIP): 02140 (G) TELEPHONE: 617 876-1170 (H) TELEFAX: 617 876-5851 (ii) TITLE OF INVENTION: BMP-10 COMPOSITIONS (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1442 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Bos taurus (B) STRAIN: bovine BMP-10 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 167..1105 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 165..778 (D) OTHER INFORMATION: /note= "partial coding sequence for propeptide" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 779..1102 (D) OTHER INFORMATION: /note= "beginning of mature peptide" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 163..164 (D) OTHER INFORMATION: /note= "3' end of intron" (ix) (D) OTHER INFORMATION: /note= "the last two-thirds of a codon which is interrupted by an intron" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTCGGTACTT CCTCTTAGAG AATGCCAACA CTGTGTTTGT TTTCACTGAT TTTCCTTCAT 60 TCTTTCTGTG TGGAGAGAAT GGACAGGCAC TCTTATTGCA TAAATAAGCA TCTGTTTTCC 120 TCTGCTACAT GCTGCAAATC TGATTTCTTT TTTGTTTTTT CCAGAT CTG TTT TCC 175 Leu Phe Ser -204 CAA CCA GCC AGT TTT AAT GGA CTC CGA AAA TAC CCT CTC CTC TTC AAC 223 Gin Pro Ala Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu Leu Phe Asn -200 -195 -190 GTA TCC ATC CCT CAC CAT GAA GAC ATC ATC ATG GCT GAG CTC AGG TTG 271 Val Ser lie Pro His His Glu Asp lie lie Met Ala Glu Leu Arg Leu -185 -180 -175 -170 TAC ACC CTG GTG CAA AGA GAC CGC CTT ATA TAT GAA GGA GTG GAC CGA 319 Tyr Thr Leu Val Gin Arg Asp Arg Leu He Tyr Glu Gly Val Asp Arg -165 -160 -155 AAA ATC ACC ATT TTT GAA GTA CTT GAG AGC AAA GAG GAC CAT GAA GGG 367 Lys He Thr He Phe Glu Val Leu Glu Ser Lys Glu Asp His Glu Gly -150 -145 -140 GAA AGA AAC ATG CTG GTC TTG GTG TCA GGG GAG ATC TAC GGA ACC AAC 415 Glu Arg Asn Met Leu Val Leu Val Ser Gly Glu He Tyr Gly Thr Asn -135 -130 -125 AGT GAG TGG GAG ACT TTT GAT GTC ACT GAT GCC ATC AGG CAT TGG CAA 463 Ser Glu Trp Glu Thr Phe Asp Val Thr Asp Ala He Arg His Trp Gin -120 -115 -110 AAG TCA GGC TCA TCC ACC CAC CAG CTG GAG GTC CAC ATT GAG AGC AAA 511 Lys Ser Gly Ser Ser Thr His Gin Leu Glu Val His He Glu Ser Lys -105 -100 -95 -90 CAC GAA ATG GAG GAC ACA CTT GGC AGG GGA CAG CTG GAA ATA GAC ACT 559 His Glu Met Glu Asp Thr Leu Gly Arg Gly Gin Leu Glu He Asp Thr -85 -80 -75 AGT GCC CGG AAT AAG CAC GAT CCT TTG CTT GTC GTG TTT TCT GAT GAC 607 Ser Ala Arg Asn Lys His Asp Pro Leu Leu Val Val Phe Ser Asp Asp -70 -65 -60 CAA AGC AGT GAG AAG GAG CGG AAA GAG GAA CTG GAT GAA ATG ATC GCC 655 Gin Ser Ser Glu Lys Glu Arg Lys Glu Glu Leu Asp Glu Met He Ala -55 -50 -45 CAC GAG CAA TTC CCA GAG ATG GAC AAC CTG GAT TTG GAC GGT TAT TCC 703 His Glu Gin Phe Pro Glu Met Asp Asn Leu Asp Leu Asp Gly Tyr Ser -40 -35 -30 AAC GGA CCT GGG GAA GAG GCT TTG CTG CAG ATG AGG TCG AAT ATC ATC 751 Asn Gly Pro Gly Glu Glu Ala Leu Leu Gin Met Arg Ser Asn He He -25 -20 -15 -10 TAT GAC TCC ACT GCC CGC ATC AGA AGG AAT GCA AAA GGA AAC TAC TGC 799 Tyr Asp Ser Thr Ala Arg He Arg Arg Asn Ala Lys Gly Asn Tyr Cys -5 1 5 AAG AGG ACC CCG CTC TAC ATC GAC TTC AAG GAG ATT GGC TGG GAC TCT 847 Lys Arg Thr Pro Leu Tyr He Asp Phe Lys Glu He Gly Trp Asp Ser 10 15 20 TGG ATC ATC GCT CCA CCT GGA TAT GAA GCC TAT GAA TGT CGT GGT GTT 895 Trp He He Ala Pro Pro Gly Tyr Glu Ala Tyr Glu Cys Arg Gly Val 25 30 35 TGC AAC TAC CCC CTG GCA GAG CAT CTC ACC CCC ACA AAG CAT GCG ATT 943 Cys Asn Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys His Ala He 40 45 50 55 ATC CAG GCC TTG GTC CAC CTC AAG AAT TCC CAG AAG GCT TCC AAA GCC 991 He Gin Ala Leu Val His Leu Lys Asn Ser Gin Lys Ala Ser Lys Ala 60 65 70 TGC TGT GTG CCC ACC AAG CTC GAG CCC ATC TCC ATC CTC TAT TTA GAT 1039 Cys Cys Val Pro Thr Lys Leu Glu Pro He Ser He Leu Tyr Leu Asp 75 80 85 AAG GGC GTC GTC ACC TAC AAG TTT AAA TAT GAG GGC ATG GCT GTC TCT 1087 Lys Gly Val Val Thr Tyr Lys Phe Lys Tyr Glu Gly Met Ala Val Ser 90 95 100 GAA TGT GGC TGT AGA TAGGAGAGGA ATCCTGTGGC TTATTTAATA ACTGTAAATG 1142 Glu Cys Gly Cys Arg 105 TGTATATTTT GTGTTCCTAT TTAATGAGAT TATTTAATAA GGGTGTACAG ATCATAGAGG 1202 CTTGCTGCCT TAGGGAATTT GACAGGTCGG TTTGTTGTAG GAAATCCATG TTTTACTCTA 1262 CAGTCGAGTC CCTTCCAATC TATTTTTCTT TGGACTTACC ATGTCCTGCA ATGCCATCTC 1322 TAACAGCAAG GCAAGCCCAC ACTACTTGCC TTCTATGTCA ATTCAAAAGG AACACCGCTA 1382 AGCAGAAATA CAGTGTCAGG AGAGGTAGAT ATTTGTGTAT GTATATGTGT ACATAGATAA 1442 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 312 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Leu Phe Ser Gin Pro Ala Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu -204 -200 -195 -190 Leu Phe Asn Val Ser He Pro His His Glu Asp He He Met Ala Glu -185 -180 -175 Leu Arg Leu Tyr Thr Leu Val Gin Arg Asp Arg Leu He Tyr Glu Gly -170 -165 -160 Val Asp Arg Lys He Thr He Phe Glu Val Leu Glu Ser Lys Glu Asp -155 -150 -145 His Glu Gly Glu Arg Asn Met Leu Val Leu Val Ser Gly Glu He Tyr -140 -135 -130 -125 Gly Thr Asn Ser Glu Trp Glu Thr Phe Asp Val Thr Asp Ala He Arg -120 -115 -110 His Trp Gin Lys Ser Gly Ser Ser Thr His Gin Leu Glu Val His He -105 -100 -95 Glu Ser Lys His Glu Met Glu Asp Thr Leu Gly Arg Gly Gin Leu Glu -90 -85 -80 He Asp Thr Ser Ala Arg Asn Lys His Asp Pro Leu Leu Val Val Phe -75 -70 -65 Ser Asp Asp Gin Ser Ser Glu Lys Glu Arg Lys Glu Glu Leu Asp Glu -60 -55 -50 -45 Met He Ala His Glu Gin Phe Pro Glu Met Asp Asn Leu Asp Leu Asp -40 -35 -30 Gly Tyr Ser Asn Gly Pro Gly Glu Glu Ala Leu Leu Gin Met Arg Ser -25 -20 -15 Asn He He Tyr Asp Ser Thr Ala Arg He Arg Arg Asn Ala Lys Gly -10 -5 1 Asn Tyr Cys Lys Arg Thr Pro Leu Tyr He Asp Phe Lys Glu He Gly 5 10 15 20 Trp Asp Ser Trp He He Ala Pro Pro Gly Tyr Glu Ala Tyr Glu Cys 25 30 35 Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys 40 45 50 His Ala He He Gin Ala Leu Val His Leu Lys Asn Ser Gin Lys Ala 55 60 65 Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro He Ser He Leu 70 75 80 Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys Tyr Glu Gly Met 85 90 95 100 Ala Val Ser Glu Cys Gly Cys Arg 105 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 197 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (B) STRAIN: Human BMP-10 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 30..167 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCTAGACCTA TGAATGTCGT GGTGTTTGC AAC TAC CCC CTG GCA GAG CAT CTC 53 Asn Tyr Pro Leu Ala Glu His Leu 1 5 ACA CCC ACA AAG CAT GCA ATT ATC CAG GCC TTG GTC CAC CTC AAG AAT 101 Thr Pro Thr Lys His Ala He He Gin Ala Leu Val His Leu Lys Asn 10 15 20 TCC CAG AAA GCT TCC AAA GCC TGC TGT GTG CCC ACA AAG CTA GAG CCC 149 Ser Gin Lys Ala Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro 25 30 35 40 ATC TCC ATC CTC TAT TTA GATAAGGGCG TCGTCACCTA CAAGGGATCC 197 He Ser He Leu Tyr Leu 45 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Asn Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys His Ala He He 1 5 10 15 Gin Ala Leu Val His Leu Lys Asn Ser Gin Lys Ala Ser Lys Ala Cys 20 25 30 Cys Val Pro Thr Lys Leu Glu Pro He Ser He Leu Tyr Leu 35 40 45 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: primer A to bovine BMP-10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TGCTCTAGAC CTATGAATGT CGTGGTGTTT GC 32 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: primer B to BMP-10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TAGGGATCCC TTGTAGGTGA CGACGCCCTT ATC 33 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: DNA inserted into pMT2 CXM (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CATGGGCAGC TCGAG 15 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Bingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: DNA inserted into pMT21 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..6 (D) OTHER INFORMATION: /note= "PstI restriction site" (ix) FEATURE: (A) NAME/KEY: misc feature (B) LOCATION: 15..56 (D) OTHER INFORMATION: /note= "Eco RI and Xhol restriction sites" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTGCAGGCGA GCCTGAATTC CTCGAGCCAT CATG 34 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 68 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Portion of the EMC virus leader sequence (x) PUBLICATION INFORMATION: (A) AUTHORS: Jung, S K (C) JOURNAL: J. Virol. (D) VOLUME: 63 (F) PAGES: 1651-1660 (G) DATE: 1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCC TTTGAAAAAC 60 ACGATTGC 68 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1584 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Human BMP-10 (vii) IMMEDIATE SOURCE: (B) CLONE: 20 GEN.3/HFL-3 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 160..1431 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 160..1107 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 1108..1431 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGGGAGAGGA AGAGTGGTAG GGGGAGGGAG AGAGAGAGGA AGAGTTTCCA AACTTGTCTC 60 CAGTGACAGG AGACATTTAC GTTCCACAAG ATAAAACTGC CACTTAGAGC CCAGGGAAGC 120 TAAACCTTCC TGGCTTGGCC TAGGAGCTCG AGCGGAGTC ATG GGC TCT CTG GTC 174 Met Gly Ser Leu Val -316-315 CTG ACA CTG TGC GCT CTT TTC TGC CTG GCA GCT TAC TTG GTT TCT GGC 222 Leu Thr Leu Cys Ala Leu Phe Cys Leu Ala Ala Tyr Leu Val Ser Gly -310 -305 -300 AGC CCC ATC ATG AAC CTA GAG CAG TCT CCT CTG GAA GAA GAT ATG TCC 270 Ser Pro He Met Asn Leu Glu Gin Ser Pro Leu Glu Glu Asp Met Ser -295 -290 -285 -280 CTC TTT GGT GAT GTT TTC TCA GAG CAA GAC GGT GTC GAC TTT AAC ACA 318 Leu Phe Gly Asp Val Phe Ser Glu Gin Asp Gly Val Asp Phe Asn Thr -275 -270 -265 CTG CTC CAG AGC ATG AAG GAT GAG TTT CTT AAG ACA CTA AAC CTC TCT 366 Leu Leu Gin Ser Met Lys Asp Glu Phe Leu Lys Thr Leu Asn Leu Ser -260 -255 -250 GAC ATC CCC ACG CAG GAT TCA GCC AAG GTG GAC CCA CCA GAG TAC ATG 414 Asp He Pro Thr Gin Asp Ser Ala Lys Val Asp Pro Pro Glu Tyr Met -245 -240 -235 TTG GAA CTC TAC AAC AAA TTT GCA ACA GAT CGG ACC TCC ATG CCC TCT 462 Leu Glu Leu Tyr Asn Lys Phe Ala Thr Asp Arg Thr Ser Met Pro Ser -230 -225 -220 GCC AAC ATC ATT AGG AGT TTC AAG AAT GAA GAT CTG TTT TCC CAG CCG 510 Ala Asn He He Arg Ser Phe Lys Asn Glu Asp Leu Phe Ser Gin Pro -215 -210 -205 -200 GTC AGT TTT AAT GGG CTC CGA AAA TAC CCC CTC CTC TTC AAT GTG TCC 558 Val Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu Leu Phe Asn Val Ser -195 -190 -185 ATT CCT CAC CAT GAA GAG GTC ATC ATG GCT GAA CTT AGG CTA TAC ACA 606 He Pro His His Glu Glu Val He Met Ala Glu Leu Arg Leu Tyr Thr -180 -175 -170 CTG GTG CAA AGG GAT CGT ATG ATA TAC GAT GGA GTA GAC CGG AAA ATT 654 Leu Val Gin Arg Asp Arg Met He Tyr Asp Gly Val Asp Arg Lys He -165 -160 -155 ACC ATT TTT GAA GTG CTG GAG AGC AAA GGG GAT AAT GAG GGA GAA AGA 702 Thr He Phe Glu Val Leu Glu Ser Lys Gly Asp Asn Glu Gly Glu Arg -150 -145 -140 AAC ATG CTG GTC TTG GTG TCT GGG GAG ATA TAT GGA ACC AAC AGT GAG 750 Asn Met Leu Val Leu Val Ser Gly Glu He Tyr Gly Thr Asn Ser Glu -135 -130 -125 -120 TGG GAG ACT TTT GAT GTC ACA GAT GCC ATC AGA CGT TGG CAA AAG TCA 798 Trp Glu Thr Phe Asp Val Thr Asp Ala He Arg Arg Trp Gin Lys Ser -115 -110 -105 GGC TCA TCC ACC CAC CAG CTG GAG GTC CAC ATT GAG AGC AAA CAC GAT 846 Gly Ser Ser Thr His Gin Leu Glu Val His He Glu Ser Lys His Asp -100 -95 -90 GAA GCT GAG GAT GCC AGC AGT GGA CGG CTA GAA ATA GAT ACC AGT GCC 894 Glu Ala Glu Asp Ala Ser Ser Gly Arg Leu Glu He Asp Thr Ser Ala -85 -80 -75 CAG AAT AAG CAT AAC CCT TTG CTC ATC GTG TTT TCT GAT GAC CAA AGC 942 Gin Asn Lys His Asn Pro Leu Leu He Val Phe Ser Asp Asp Gin Ser -70 -65 -60 AGT GAC AAG GAG AGG AAG GAG GAA CTG AAT GAA ATG ATT TCC CAT GAG 990 Ser Asp Lys Glu Arg Lys Glu Glu Leu Asn Glu Met He Ser His Glu -55 -50 -45 -40 CAA CTT CCA GAG CTG GAC AAC TTG GGC CTG GAT AGC TTT TCC AGT GGA 1038 Gin Leu Pro Glu Leu Asp Asn Leu Gly Leu Asp Ser Phe Ser Ser Gly -35 -30 -25 CCT GGG GAA GAG GCT TTG TTG CAG ATG AGA TCA AAC ATC ATC TAT GAC 1086 Pro Gly Glu Glu Ala Leu Leu Gin Met Arg Ser Asn He He Tyr Asp -20 -15 -10 TCC ACT GCC CGA ATC AGA AGG AAC GCC AAA GGA AAC TAC TGT AAG AGG 1134 Ser Thr Ala Arg He Arg Arg Asn Ala Lys Gly Asn Tyr Cys Lys Arg -5 1 5 ACC CCG CTC TAC ATC GAC TTC AAG GAG ATT GGG TGG GAC TCC TGG ATC 1182 Thr Pro Leu Tyr He Asp Phe Lys Glu He Gly Trp Asp Ser Trp He 10 15 20 25 ATC GCT CCG CCT GGA TAC GAA GCC TAT GAA TGC CGT GGT GTT TGT AAC 1230 He Ala Pro Pro Gly Tyr Glu Ala Tyr Glu Cys Arg Gly Val Cys Asn 30 35 40 TAC CCC CTG GCA GAG CAT CTC ACA CCC ACA AAG CAT GCA ATT ATC CAG 1278 Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys His Ala He He Gin 45 50 55 GCC TTG GTC CAC CTC AAG AAT TCC CAG AAA GCT TCC AAA GCC TGC TGT 1326 Ala Leu Val His Leu Lys Asn Ser Gin Lys Ala Ser Lys Ala Cys Cys 60 65 70 GTG CCC ACA AAG CTA GAG CCC ATC TCC ATC CTC TAT TTA GAC AAA GGC 1374 Val Pro Thr Lys Leu Glu Pro He Ser He Leu Tyr Leu Asp Lys Gly 75 80 85 GTC GTC ACC TAC AAG TTT AAA TAC GAA GGC ATG GCC GTC TCC GAA TGT 1422 Val Val Thr Tyr Lys Phe Lys Tyr Glu Gly Met Ala Val Ser Glu Cys 90 95 100 105 GGC TGT AGA TAGAAGAAGA GTCCTATGGC TTATTTAATA ACTGTAAATG 1471 Gly Cys Arg TGTATATTTG GTGTTCCTAT TTAATGAGAT TATTTAATAA GGGTGTACAG TAATAGAGGC 1531 TTGCTGCCTT CAGGAAATGG ACAGGTCAGT TTGTTGTAGG AAATGCATAT TTT 1584 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 424 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Gly Ser Leu Val Leu Thr Leu Cys Ala Leu Phe Cys Leu Ala Ala -316 -315 -310 -305 Tyr Leu Val Ser Gly Ser Pro He Met Asn Leu Glu Gin Ser Pro Leu -300 -295 -290 -285 Glu Glu Asp Met Ser Leu Phe Gly Asp Val Phe Ser Glu Gin Asp Gly -280 -275 -270 Val Asp Phe Asn Thr Leu Leu Gin Ser Met Lys Asp Glu Phe Leu Lys -265 -260 -255 Thr Leu Asn Leu Ser Asp He Pro Thr Gin Asp Ser Ala Lys Val Asp -250 -245 -240 Pro Pro Glu Tyr Met Leu Glu Leu Tyr Asn Lys Phe Ala Thr Asp Arg -235 -230 -225 Thr Ser Met Pro Ser Ala Asn He He Arg Ser Phe Lys Asn Glu Asp -220 -215 -210 -205 Leu Phe Ser Gin Pro Val Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu -200 -195 -190 Leu Phe Asn Val Ser He Pro His His Glu Glu Val He Met Ala Glu -185 -180 -175 Leu Arg Leu Tyr Thr Leu Val Gin Arg Asp Arg Met He Tyr Asp Gly -170 -165 -160 Val Asp Arg Lys He Thr He Phe Glu Val Leu Glu Ser Lys Gly Asp -155 -150 -145 Asn Glu Gly Glu Arg Asn Met Leu Val Leu Val Ser Gly Glu He Tyr -140 -135 -130 -125 Gly Thr Asn Ser Glu Trp Glu Thr Phe Asp Val Thr Asp Ala He Arg -120 -115 -110 Arg Trp Gin Lys Ser Gly Ser Ser Thr His Gin Leu Glu Val His He -105 -100 -95 Glu Ser Lys His Asp Glu Ala Glu Asp Ala Ser Ser Gly Arg Leu Glu -90 -85 -80 He Asp Thr Ser Ala Gin Asn Lys His Asn Pro Leu Leu He Val Phe -75 -70 -65 Ser Asp Asp Gin Ser Ser Asp Lys Glu Arg Lys Glu Glu Leu Asn Glu -60 -55 -50 -45 Met He Ser His Glu Gin Leu Pro Glu Leu Asp Asn Leu Gly Leu Asp -40 -35 -30 Ser Phe Ser Ser Gly Pro Gly Glu Glu Ala Leu Leu Gin Met Arg Ser -25 -20 -15 Asn He He Tyr Asp Ser Thr Ala Arg He Arg Arg Asn Ala Lys Gly -10 -5 1 Asn Tyr Cys Lys Arg Thr Pro Leu Tyr He Asp Phe Lys Glu He Gly 5 10 15 20 Trp Asp Ser Trp He He Ala Pro Pro Gly Tyr Glu Ala Tyr Glu Cys 25 30 35 Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys 40 45 50 His Ala He He Gin Ala Leu Val His Leu Lys Asn Ser Gin Lys Ala 55 60 65 Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro He Ser He Leu 70 75 80 Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys Tyr Glu Gly Met 85 90 95 100 Ala Val Ser Glu Cys Gly Cys Arg 105

Claims: What is claimed is: 1. An isolated DNA sequence encoding BMP-10 protein. 2. The DNA sequence of claim 1 wherein said DNA sequence is selected from the group consisting of: (a) nucleotides #779 or #797 to #1102 of SEQ ID NO: 1; (b) nucleotides #1108 or #1126 to #1431 of SEQ ID NO: 10; and (c) sequences which hybridize to (a) or (b) under stringent hybridization conditions and encode a protein which exhibits the ability to form cartilage and/or bone. 3. The DNA sequence of claim 1 wherein said DNA sequence is selected from the group consisting of: (a) nucleotides encoding for amino acids # 1 to #108 of SEQ ID NO: 2; (b) nucleotides encoding for amino acids #1 to #108 of SEQ ID NO:11; and (c) sequences which hybridize to (a) or (b) under stringent hybridization conditions and encode a protein which exhibits the ability to form cartilage and/or bone. 4. A host cell transformed with the DNA sequence of claim 1. 5. A host cell transformed with the DNA sequence of claim 2. 6. A host cell transformed with the DNA sequence of claim 3. 7. An isolated DNA molecule having a sequence encoding a protein which is characterized by the ability to induce the formation of cartilage and/or bone, said DNA molecule comprising a DNA sequence selected from the group consisting of: (a) nucleotide # 779 to # 1105 of SEQ ID NO: 1; and (b) nucleotide # 1108 to # 1431 of SEQ ID NO: 10; and (c) naturally occurring allelic sequences and equivalent degenerative codon sequences of (a) or (b) . 8. A host cell transformed with the DNA molecule of claim 7. 9. A vector comprising a DNA molecule of claim 7 in operative association with an expression control sequence therefor . 10. A host cell transformed with the vector of claim 9. 11. An isolated DNA molecule encoding a BMP-10 protein, said DNA molecule comprising nucleotide # 160 to # 1431 of SEQ ID NO: 10. 12. An isolated DNA molecule comprising a DNA coding sequence encoding a BMP-10 protein, said DNA molecule comprising nucleotides # 1108 to # 1431 of SEQ ID NO:10, and further comprising a nucleotide sequence encoding a suitable propeptide 5' to and linked in frame to the DNA coding sequence. 13. A vector comprising a DNA molecule of claim 11 in operative association with an expression control sequence therefor. 14. A host cell transformed with the vector of claim 12. 15. A method for producing a purified BMP-10 protein said method comprising the steps of: (a) culturing a host cell transformed with a DNA sequence according to claim 2 , comprising a nucleotide sequence encoding a BMP-10 protein; and (b) recovering and purifying said BMP-10 protein from the culture medium. 16. A method for producing a purified BMP-10 protein said method comprising the steps of: (a) culturing a host cell transformed with a DNA sequence according to claim 3, comprising a nucleotide sequence encoding a BMP-10 protein; and (b) recovering and purifying said BMP-10 protein from the culture medium. 17. A method for producing a purified BMP-10 protein said method comprising the steps of: (a) culturing a host cell transformed with a DNA sequence according to claim 7, comprising a nucleotide sequence encoding a BMP-10 protein; and (b) recovering and purifying said BMP-10 protein from the culture medium. 18. A purified BMP-10 polypeptide comprising the amino acid sequence from amino acid #1 to amino acid #108 as set forth in SEQ ID NO: 11. 19. A purified BMP-IO polypeptide of claim 18 wherein said polypeptide is a dimer wherein each subunit comprises at least the amino acid sequence from amino acid #1 to amino acid #108 of SEQ ID NO:11. 20. A purified BMP-10 polypeptide of claim 18, wherein said polypeptide is a dimer wherein one subunit comprises at least the amino acid sequence from amino acid #1 to amino acid #108 of SEQ ID NO:11, and one subunit comprises an amino acid sequence for a bone morphogenetic protein selected from the group consisting BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP- 7, BMP-8 and BMP-9. 21. A purified BMP-10 protein produced by the steps of (a) culturing a cell transformed with a DNA comprising the nucleotide sequence from nucleotide #1108 to #1431 as shown in SEQ ID NO:10; and (b) recovering and purifying from said culture medium a protein comprising the amino acid sequence from amino acid #1 to amino acid #108 as shown in SEQ ID NO:11. 22. The purified BMP-10 protein of claim 21, wherein the cell is a mammalian cell, and the DNA additionally comprises nucleotides #160 to #1107 of SEQ ID NO:10. 23. A purified BMP-10 protein characterized by the ability to induce the formation of cartilage and/or bone. 24. A pharmaceutical composition comprising an effective amount of the BMP-10 protein of claim 23 in admixture with a pharmaceutically acceptable vehicle. 25. A composition of claim 24 further comprising a matrix for supporting said composition and providing a surface for bone and/or cartilage growth. 26. The composition of claim 25 wherein said matrix comprises a material selected from the group consisting of hydroxyapatite, collagen, polylactic acid and tricalcium phosphate. 27. A method for inducing bone and/or cartilage formation in a patient in need of same comprising administering to said patient an effective amount of the composition of claim 25. 45 SUBSTITUTE SHEET RULE 25 28. A chimeric DNA molecule comprising a DNA sequence encoding a propeptide from a member of the TGF-/3 superfamily of proteins linked in correct reading frame to a DNA sequence encoding a BMP-10 polypeptide.

Inventors: CELESTE ANTHONY J, WOZNEY JOHN M, CELESTE, ANTHONY, J., WOZNEY, JOHN, M.
Applicants: GENETICS INST, GENETICS INSTITUTE, INC.
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, A61P43, C07H21, C07K14, C12N5, C12N15, C12P21, C12R1